Degenerated PCR Primer L14912-CYB, H115149-CYB (Fish), amplify ~285 pb, Minamoto et al. 2012

PCR #1 May/15/2019. Pag 19

PCR reaction

  • 30 ng/ul DNA aprox
  • 1 ul primer (10Mm)
  • Biomix 12.5 ul
  • 25 ul total reaction

SAMPLES

Sample Volume (ul)
IPT 1 0.7
IPT 2 0.5
IPT 3 0.5
IPT 4 0.5
IPT 5 0.5
IPT 6 1.2
GSO 1 0.7
GSO 2 1.2
GSO 3 0.5
GSO 4 0.7
GSO 5 1
GSO 6 1
GSO 7 1.5
G 2 1

*1 NEGATIVE

Cycling conditions (Minamoto et al. 2012)

  • 2 min to 94°C
  • 35 cycles: 94°C 30 s, 50°C 30 s, 72°C 30 s,
  • 10 min to 72°C
  • 4°C forever
PCR CYB May/15/2019

PCR CYB May/15/2019

1% gel, GelGreen, 1:30 hour at 100v

The samples didn´t amplify, the next test I will use Prada´s cycling condition and 40°C annealing temperature.

PCR #2 May/20/2019. Pag 22

PCR reaction

  • 30 ng/ul DNA
  • 0.5 ul primer (10Mm)
  • Biomix 12.5 ul
  • 25 ul total reaction

SAMPLES

IPT 1, IPT 2, GSO 1, GSO 2, E 1, E 2, A 1 (1 NEG)

Prada´s Cycling conditions

  • 2 min to 94°C
  • 90 s 50°C
  • 2 min to 72°C
  • 36 cycles: 94°C for 45 s, 48°C for 1 min, 72°C for 90 s,
  • 1 cycle: 94°C for 40 s, 50°C for 1 min, 72°C for 10 min,
  • 12°C forever

I tried: primer 0.5 ul and new cycling condition (Prada). I used samples from Indian Point (IPT), Graduate School of Oceanografphy Beach (GSO) and old extractions.

PCR #3 May/20/2019. Pag 23

PCR reaction

  • 30 ng/ul DNA
  • 1 ul primer (10Mm)
  • Biomix 12.5 ul
  • 25 ul total reaction

SAMPLES

E 1, E 2, A 1, D 2, A 3, I 2, G 1 (1 NEG)

I used Prada`s Cycling conditions and just used old extractions

PCR CYB May/20/2019. Pag 22 y 23

PCR CYB May/20/2019. Pag 22 y 23

1% gel, GelGreen, 1:30 hour at 100v

With primer 0.5 ul. Just old extraction amplified (Pag 22)

With primer 1 ul (Pag 23).

PCR #4 May/29/2019. Pag 31

PCR reaction

  • 30 ng/ul DNA aprox
  • 1 ul primer (10Mm)
  • Biomix 12.5 ul
  • 25 ul total reaction
  • 2 ul BSA

SAMPLES

Sample Extraction
D 2 Old extraction
E 1 Old extraction
1B GLfilter, ZYMO KIT
3B GL filter, ZYMO KIT
5B GL filter, ZYMO KIT
2B CP filter, ZYMO KIT
4B CP filter, ZYMO KIT
6B CP filter, ZYMO KIT
1A GL filter, QIAGEN KIT
3A GL filter, QIAGEN KIT
5A GL filter, QIAGEN KIT
2A CP filter, QIAGEN KIT
4A CP filter, QIAGEN KIT
6A CP filter, QIAGEN KIT
IPT 1 (30) Indian Point extraction
IPT 2 (30) Indian Point extraction
IPT 3 (30) Indian Point extraction
GSO 1 (30) Graduate School of Oceanografphy Beach
GSO 2 (30) Graduate School of Oceanografphy Beach
GSO 3 (30) Graduate School of Oceanografphy Beach
GSO 4 (30) Graduate School of Oceanografphy Beach

*1 NEGATIVE

I test Prada’s Cycling conditions (50°C), BSA, two differents filters and two differents extraction kits

PCR CYB May/29/2019. Pag 31

PCR CYB May/29/2019. Pag 31

1% gel, GelGreen, 1:30 hour at 100v

- Just amplified one new extraction 5A (GL filter, QIAGEN KIT),

- I will try 48°C annealing again, 20 ng/ul DNA template, 1.5 ul BSA.

PCR #5 May/31/2019. Pag 33

PCR reaction

  • 20 ng/ul DNA aprox
  • 1 ul primer (10Mm)
  • 12.5 ul Biomix
  • 25 ul total reaction
  • 1.5 ul BSA

SAMPLES

Sample Extraction
E 1 Old extraction
1B GLfilter, ZYMO KIT
2B CP filter, ZYMO KIT
1A GL filter, QIAGEN KIT
2A CP filter, QIAGEN KIT
IPT 1 (30) Indian Poin extraction
GSO 1 (30) Graduate School of Oceanografphy Beach
  • 1 NEGATIVE

I test Prada’s Cycling conditions (48°C), 1.5 ul BSA, 20 ng/ul DNA template

PCR CYB May/31/2019. Pag 33

PCR CYB May/31/2019. Pag 33

1% gel, GelGreen, 1:30 hour at 100v

- Contamination :(

- I will try another samples and new H20.

PCR #6 Jun/12/2019. Pag 34

PCR reaction

  • 20 ng/ul DNA aprox
  • 1 ul primer (10Mm)
  • 12.5 ul Biomix
  • 25 ul total reaction
  • 1.5 ul BSA

SAMPLES

Sample Extraction
E 1 Old extraction
3B GLfilter, ZYMO KIT
4B CP filter, ZYMO KIT
3A GL filter, QIAGEN KIT
4A CP filter, QIAGEN KIT
IPT 2 (30) Indian Poin extraction
GSO 2 (30) Graduate School of Oceanografphy Beach
  • 1 NEGATIVE

I test Carlos’ Cycling conditions (48°C), new H20, new samples

PCR CYB Jun/12/2019. Pag 34 1% gel, GelGreen, 1:30 hour at 100v

- Contamination :(

- I will use new reagents.

PCR #7 Jun/12/2019. Pag 35

PCR reaction

  • 20 ng/ul DNA aprox
  • 1 ul primer (10Mm)
  • 12.5 ul Biomix
  • 25 ul total reaction
  • 1.5 ul BSA

SAMPLES

Sample Extraction
E 1 Old extraction
3B GLfilter, ZYMO KIT
4B CP filter, ZYMO KIT
3A GL filter, QIAGEN KIT
4A CP filter, QIAGEN KIT
IPT 2 (30) Indian Poin extraction
GSO 2 (30) Graduate School of Oceanografphy Beach
  • 1 NEGATIVE

Prada’s Cycling conditions (48°C), new reagents

PCR CYB Jun/13/2019. Pag 34

PCR CYB Jun/13/2019. Pag 34

1% gel, GelGreen, 1:30 hour at 100v

- Contamination :(